Intraosseous administration into the skull: Potential blood–brain barrier bypassing route for brain drug delivery

Abstract Progress in treating central nervous system (CNS) disorders is retarded owing to a limited understanding of brain disease pathology. Additionally, the blood–brain barrier (BBB) limits molecular entry into the brain. Many approaches for brain drug delivery to overcome the BBB, such as BBB permeability enhancement, transient BBB disruption, and direct surgical administration have been explored with limited success. Recent research has shown that direct vascular channels exist between the skull bone marrow and the meninges, allowing myeloid and lymphoid cells to migrate. We hypothesized that these direct channels may also allow brain drug delivery from the skull bone marrow to the brain. In this study, for the first time we propose intraosseous administration of drugs into the skull (intracalvariosseous [ICO]) as a novel approach for brain drug delivery via BBB bypassing routes. We tested the feasibility of the approach by applying nine representative compounds over thinned mouse skulls to simulate ICO and measuring the compound entry level in the brain compared to that after systemic administration. Surprisingly, we found that the skull is not completely impermeable to drug penetration into the brain and the tested compounds reached the brain tissue several tens‐to‐hundred times higher by ICO than systemic application. These findings suggest a role for the BBB bypassing route from skull to brain, apart from the systemic route, in the drug entry into the brain after ICO. This approach should be applicable to other CNS drugs and even BBB impermeable drugs. Overall ICO provides an innovative and advantageous pathway for effective treatment of brain diseases.

or no understanding of the relevant pathophysiology of CNS disorders, difficulties in developing preclinical models and assessing target engagement, the presence of the blood-brain barrier (BBB), CNS-mediated side effects, the lack of clinical scales of sensitivity, and interference with the therapeutic benefit of placebo effects. [4][5][6] The major obstacle to drug entry into the brain is the presence of the BBB, composed of endothelial cells linking tight junctions. 7 The BBB, with its distinctive structure, maintains homeostasis by regulating efflux and influx, and protects the brain from pathogenic agents. 8 To overcome the BBB, approaches such as enhancement of BBB permeability, transient BBB disruption, and local direct administration by surgery have long been on the list of possible clinical practices. 9,10 Various strategies involving the design and modification of active drug molecules and nanomaterial-based drug delivery have long been studied to enhance BBB permeability. In addition, the transient opening of tight junctions using hyperosmotic solutions, focused ultrasound, and electromagnetic fields can potentially permit the entry of CNS drugs into the brain in efficacious amounts. 10 Although significant progress has been made in overcoming the BBB to deliver CNS drugs to the brain, many approaches have provided limited clinical success.
Recent findings demonstrate the existence of direct ossified vascular channels between the skull bone marrow and meninges, allowing myeloid and lymphoid cells to migrate. [11][12][13][14][15] Research has shown the presence of hundreds of capillaries known as trans-cortical vessels (TCVs), which form a direct connection between endosteal and periosteal circulation. Furthermore, structures similar to TCVs in flat calvaria bone were identified, thus confirming the presence of direct channels between the bone marrow and brain surface. 11,16 Additionally, it was demonstrated that cerebrospinal fluid (CSF) enters skull bone marrow niches in physiology and pathology, where it modulates myelopoiesis and egress to meninges. 17 We speculated that these channels could be bi-directional, enabling bone marrow to have direct access to the CSF.
Furthermore, we hypothesized that these direct channels may also allow drug delivery from the skull bone marrow to the brain.
In this study, we propose for the first time, the intraosseous administration of CNS drugs into the skull (intracalvariosseous [ICO]) as a novel approach for brain drug delivery via BBB-bypassing routes.
Our goal was to verify the clinical feasibility and evaluate the comparative advantage of ICO over systemic administration using quantitative pharmacokinetic analysis. We expected that the highly porous diploes and permeable cortex of the skull bone 18 would allow CNS drugs applied into the diploes to cross the cortex to the brain parenchyma. Brain drug delivery through the ICO administration route is completely different from various attempts at overcoming the BBB.

| Animals
All animal procedures were approved by the Institutional Animal Care and Use Committee at Gachon University, Republic of Korea, and complied with the guideline for users of Animal Ethics Committee of Gachon University (approval number LCDI-2020-0062).
The experiment was conducted with male BALB/c mice aged 5 weeks with a weight range of 20-25 g. To ensure statistical independence, only male mice were used for each experiment. All the experimental procedures were performed from the next day of randomization.

| Skull thinning for intraosseous administration into the skull (ICO)
To accomplish intraosseous administration into the skull (ICO administration) for evaluation of brain drug delivery, we performed

| Data analysis
To compare the results of the nine compounds (CPZ, RIS, SUC, TMZ, PTX, DPZ, RVG, GABA, and GSH) according to administration route, the compound concentrations in plasma and ISF dialysate samples were determined using an LC-MS/MS analysis and plotted over time.
The area under the curve in plasma concentration-time curves (AUC plasma ) and brain ISF concentration-time curves (AUC ISF ) were determined by non-compartmental analysis using WinNonlin 2.1 (Pharsight, Mountain View, CA). The compound concentration in the whole brain at 24 h (C br ) was determined using LC-MS/MS and then corrected for the plasma volume of the corresponding organ using equation: where C br,quant,24h = brain concentration (ng/g) at 24 h quantified by LC-MS/MS, V 0 = plasma volume of the corresponding organ (μl/g), C plasma,24h = plasma concentration (ng/ml) at 24 h. The following V 0 value for the brain was chosen as 9.3 ± 1.1 μl/g. 19 The extent of brain uptake after ICO administration as compared to IV administration was also expressed as brain availability (F br ), analogous to systemic bioavailability (F), and calculated as a ratio of AUC ISF,IV and AUC ISF,ICO using the equation: where AUC ISF,ICO and AUC ISF,IV are the area under the curve to 24 h in the brain ISF-time plot (AUC ISF ) after ICO and IV administration, respectively.

| Statistical analysis
All data are presented as mean ± SEM. Each value represents the mean of four separate experiments for each group. Significant differences were analyzed using a two-sample Student's t-test. Statistical significance was indicated by *p < 0.05 and **p < 0.01.

| Fabrication of ICO device and animal preparation for ICO administration in mice
To actualize the ICO administration in the mice, we designed the ICO device for the mice (Figure 1a,b). ICO device is a one-body-type that consists of a cylindrical reservoir with a diameter of 5 mm and a skullembedding tube with an ID of 0.8 mm, OD of 1.3 mm, and a height of 0.5 mm. The light microscopy images ( Figure S1) show that the ICO device was well fabricated with a flat surface with a surface roughness average of less than 25 μm enough to be seated into the thinned skull at 200 μm depth. It indicates that the ICO device is not inferior for embedding into the calvarial diploe, which is sponge cancellous bone, as 0.2 mm. The ICO device was sterilized in ethanol and applied to a thinned area on the mouse skull for ICO administration (Figure 1c).
The flow chart for in vivo experimental design is shown in Figure 2a.
The details of the thinned skull area and guide cannula place for insertion of microdialysis tube on the mouse skull were presented in The schematic diagram of microdialysis and ICO administration was presented in Figure 2c by coronal section view. Figure 2d shows a real mouse photo after embedding the ICO device.

| Integrity test for ICO device embedded in thinned-mouse skull by NIR fluorescence imaging with ICG
To verify the integrity of ICO administration, we applied indocyanine green (ICG) into the ICO device embedded in a thinned-mouse skull.
When we observed ICG fluorescence signals in the skull with an NIR camera after 24 h post-ICO administration, the ICG signals shone brightly at the left parietal bone where the ICO device was embedded, whereas no signal was observed at the other skull bones. In addition, no leak from the glue-sealed area was observed (Figure 3a). This clearly indicated that the ICG was diffused into the diploic space of the left parietal bone, where the ICO device was embedded.
It was estimated that the extent of drug partitioning from the ICO device to diploe within a 24 h period was approximately 10% of the device-loading dose from the integrity test with ICG ( Figure S2) and then 10-times of the IV dose was applied to the ICO in further pharmacokinetic study.

| Pharmacokinetic and in vivo brain uptake study after IV and ICO administration
To evaluate the comparative advantage for brain drug delivery of the  Table 1 summarizes the pharmacokinetic parameters of nine compounds after IV and ICO administration, including AUC plasma , AUC ISF , C br , brain/plasma ratio at 24 h (K p ), and in vivo brainavailability (F br ).
Statistical analysis of seven compounds (SUC, RIS, DPZ, RVG, TMZ, PTX, and GABA) revealed the same tendency that AUC plasma of ICO groups was relatively lower than IV groups whereas AUC ISF of ICO groups was relatively higher than IV groups. CPZ and GSH showed that AUC plasma of ICO groups was relatively lower than IV groups whereas AUC ISF of ICO groups was no different from IV groups. The AUC ratio (%, AUC ISF /AUC plasma ) revealed that ICO groups were significantly higher than IV groups, ranging from 3.0-fold (TMZ) to 156.9-fold (DPZ), except PTX and GSH (Figure 3b-j). The brain availability (F br ), analogous to systemic bioavailability (F), was calculated as a ratio of AUC ISF,IV and AUC ISF,ICO and is represented in Table 1. In particular, in the case of PTX, ISF concentration at all-time points after IV administration was under the limit of detection (LOD), resulting in to be not analyzed (NA), whereas that after ICO administration was detected, resulting in to be calculated as an AUC ISF of 796.55 ng/min/ml (Figure 3h). It is meaningful from the point of view that PTX, which could not sufficiently enter the brain to quantify after systemic administration, could do that after ICO administration.
Interestingly, the Cbr, which is a parameter representing actual brain drug concentration at 24 h, in all compounds showed that ICO groups were significantly higher than IV groups by as much as . This indicates that brain accumulation following ICO administration might be handled differently compared to IV administration, which relies only on the BBB passing route for brain drug delivery.

| Relationship between the in vivo brain uptake of compounds versus in vitro BBB permeability, molecular weight, and octanol-water partition coefficients
To investigate the relationship between the in vivo brain uptake of compounds and molecular characteristics, we evaluated the in vitro brain permeability of chosen compounds ( Figure S12). CPZ, RIS, DPZ, RVG, and TMZ selected as representatives among CNS drugs showed a PS value of 10 or higher, indicating that they can be permeable to the brain compared to the control SUC which is representative of brain impermeable compound. In particular, GABA, a neurotransmitter and biological product for neuroprotective, showed the highest PS value of 26.89 ± 1.89, whereas PTX, a widely used anticancer agent but not used for brain tumors, showed the lowest PS value of 1.74 ± 0.04.

| DISCUSSION
In this study, our data suggest that intraosseous administration into the skull (ICO) can be a novel approach for brain drug delivery of CNS drugs via BBB-bypassing routes. Importantly, the brain concentration of the nine representative compounds after ICO administration was higher (minimum 2-fold to maximum 342-fold) than that after IV administration, suggesting that ICO administration might provide a special condition in which compounds have an opportunity to be taken up into the brain.
In our findings, the regression line in the correlation analysis showed an opposite slope direction between ICO and IV administration in the whole brain concentration (C br ) and brain/plasma ratio at 24 h (K p ) related to molecular characteristics ( Figure 4). In the case of IV injection directly into the blood stream, the drugs should pass through the BBB to enter the brain, resulting in the brain concentration being influenced by the blood concentration of drugs. It has been reported that hydrophilic compounds have limited passive transport of drug molecules through the BBB, whereas lipophilic drugs smaller than 400-600 Da may enter the brain with comparative ease. 20 This is consistent with our results in the IV correlation data, showing that the lower the PS values, the lower the C br , whereas a lower MW and log Pow lead to a higher C br . However, in the case of ICO administration into the skull diploe, which showed opposite results to those with IV administration, it can be expected that the brain uptake of compounds after ICO administration is processed through a different route compared to the systemic route, which is only related to the blood-to-brain route. We expected that the transportation of molecules from the diploe to the brain would occur through the abovementioned transcranial route. The skull-to-brain (transcranial) route may involve direct channels and non-direct pathways ( Figure 5).
The meninges consist of three layers, including the dura mater, the arachnoid mater, and pia mater, that surround the brain and spinal cord. 21,22 The arachnoid barrier cell layer is distinguished by continuous and complex tight junctions, forming an epithelial bloodcerebrospinal fluid (BCSFB) between the dura mater and the subarachnoid space. [21][22][23] The CSF was filled in the subarachnoid space below the arachnoid barrier. The pia mater is a thin and transparent layer for CSF separation from the brain. 24 The pia mater has no tight junctions, 21 thus molecules can get the opportunity to easily enter the brain if they reach the CSF. It was reported that CSF accesses skull bone marrow via direct channels. 17 These channels could enable bone marrow's direct access to the CSF, allowing a route for drug delivery from the skull to the brain. Although the meninges, providing a CNS barrier, might be one impediment to the transport of drugs from the skull to the brain by ICO administration, our results strongly showed that the skull is not completely impermeable to drug penetration into the brain. The details of the drug delivery process by ICO through the transcranial route are currently unknown; it would be an interesting study to closely explore the function of the direct channel in view of the drug delivery mechanism.
To administer BBB-impermeable CNS drugs for the treatment of Compound Administration route AUC plasma (ng/min/ml) AUC ISF (ng/min/ml) C br (ng/ml) (AUC ISF,ICO /  AUC ISF,IV ) systemic route. 26,27 Nevertheless, the approaches still have limitations, such as invasive procedures and associated complications. [28][29][30] In light of these limitations related to current attempts for brain delivery of BBB-impermeable CNS drugs, ICO administration could be a potential alternative technique for brain drug delivery to address these concerns.
This study presents a new administration technique using the skull for brain drug delivery in mice. The parietal bone, which is the largest in the mouse skull (length 4.8 mm, width 31   The professional medical surgery under anesthesia-the skull thinning and device mounting-would be needed for clinical application of ICO but expected to be minimally invasive and a one-time procedure.
Although it is too early to provide any plausible predictions on the future of clinical application, ICO could find it more useful for the treatment of chronic neurological diseases than acute conditions such as strokes and head injuries. Furthermore, ICO could probably provide a less invasive alternative to the current IT devices after further optimization of many technical details.
Interestingly, in this study, we found that intraosseous administration into the skull readily delivered drugs into the brain, probably via the BBB-bypassing route. This approach could be applied to other

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

F I G U R E 5
The key transcranial route of molecules from the skull cortex penetration to the brain following ICO administration.
Molecules are transported to the brain through not only the systemic route but also the transcranial route involving direct channel and nondirect.